Antioxidant sensor

ABSTRACT

The present invention relates to a device and method for measuring the level of an oxidant or antioxidant analyte in a fluid sample. The device comprises a disposable electrochemical cell, such as a thin layer electrochemical cell, containing a reagent capable of undergoing a redox reaction with the analyte. When the device or method is to be used with slow-reacting analytes, heat may be applied to the sample by a resistive heating element in the device or by an exothermic material contained within the electrochemical cell. Application of heat will accelerate the rate of the redox reaction between the reagent and the analyte and thus facilitate the electrochemical measurement of slow-reacting analytes.

RELATED APPLICATIONS

[0001] This application is a continuation of copending application Ser.No. 09/615,691, filed on Jul. 14, 2000, which is a continuation-in-part,under 35 U.S.C. § 120, of copending International Patent Application No.PCT/AU99/00152, filed on Mar. 11, 1999 under the Patent CooperationTreaty (PCT), which was published by the International Bureau in Englishon Sep. 16, 1999, which designates the U.S. and claims the benefit ofAustralian Provisional Patent Application No. PP 2388, filed Mar. 12,1998. Application Ser. No. 09/615,691 is also a continuation-in-part ofapplication Ser. No. 09/314,251, filed May 18, 1999, now U.S. Pat. No.6,174,420. Application Ser. No. 09/314,251 is a continuation ofapplication Ser. No. 08/852,804, filed May 7, 1997, now U.S. Pat. No.5,942,102, and a continuation of application Ser. No. 09/068,828, filedMar. 15, 1999, now U.S. Pat. No. 6,179,979. Application Ser. No.08/852,804 is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/AU96/00723 which has an Internationalfiling date of Nov. 15, 1996, which designated the United States ofAmerica, and which was published by the International Bureau in Englishon May 22, 1997, and claims the benefit of Australian Provisional PatentApplication No. PN 6619, filed Nov. 16, 1995. Application Ser. No.09/068,828 is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/AU96/00724 which has an Internationalfiling date of Nov. 15, 1996, which designated the United States ofAmerica, and which was published by the International Bureau in Englishon May 22, 1997, and claims the benefit of Australian Provisional PatentApplication No. PN 6619, filed Nov. 16, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to a device and method formeasuring the level of an oxidant or antioxidant analyte in a fluidsample. The device comprises a disposable electrochemical cellcontaining a reagent capable of directly undergoing a redox reactionwith the analyte.

BACKGROUND OF THE INVENTION

[0003] An oxidation reaction, broadly defined, involves the transfer ofone or more electrons from one molecule or atom (the reducing agent orreductant) to another (the oxidizing agent or oxidant). Oxidationreactions occur in a broad range of systems, e.g., food products, livingorganisms, and drinking water, and may be detrimental or beneficial.Food products exposed to oxygen may undergo oxidative degradation,resulting in the generation of undesirable flavors and odors, thedestruction of fat-soluble vitamins and essential fatty acids, and theproduction of toxic degradation products. Beneficial oxidation reactionsin food products include those between natural or synthetic antioxidantsand oxidants, whereby the oxidant is prevented from participating in adetrimental oxidation reaction.

[0004] Thus, it is desirable to be able to measure oxidant orantioxidant levels in liquid samples in many fields. For example, it isdesirable in terms of manufacturing quality control as well as healthmonitoring to measure the level of preservatives such as sulfur dioxidein wine or food, the level of ascorbic acid in fruit, vegetables,beverages, and biological fluids, and the level of chlorine or peroxidesin water. Most conveniently, these tests are fast and easy to use and beamenable to field as well as laboratory use.

[0005] Existing methods for measuring these components require eitherexpensive laboratory apparatus or skilled operators in order for themethod to be used successfully. For example, a sensor for detectingantioxidant agents in oil is disclosed in U.S. Pat. No. 5,518,590.However, this sensor is not designed for single, disposable use and doesnot use a redox agent. It is therefore desirable to have a sensordesigned for single, disposable use that can detect oxidant orantioxidant levels in fluid samples through the use of a redox reagent.

SUMMARY OF THE INVENTION

[0006] The present invention provides a device and method for measuringoxidant and antioxidant analytes with a disposable sensing element,suitable for a single use, that can be combined with a meter to give arobust, fast, and easy to use test that is amenable to field as well aslaboratory use. In particular, the invention relates to the use of anelectrochemical sensor that utilizes a redox agent that reacts with theanalyte of interest to produce an electrochemically detectable signal.

[0007] In one embodiment of the present invention, a device fordetecting a presence or an absence of a redox reactive analyte in anaqueous sample is provided, the device including an electrochemical cellhaving a sensing chamber, a first electrode, a second electrode, anaperture for admitting the sample into the sensing chamber, and areagent contained within the sensing chamber, wherein theelectrochemical cell is designed to be disposed of after use in a singleexperiment, and wherein the reagent is capable of undergoing a redoxreaction directly with the analyte to generate an electrical signalindicative of the presence or absence of the analyte.

[0008] In one aspect of this embodiment, the first electrode is asensing electrode that may consist of platinum, palladium, carbon,indium oxide, tin oxide, gold, iridium, copper, steel, or mixturesthereof. The first electrode may also be silver. The first electrode maybe formed by a technique such as sputtering, vapor coating, screenprinting, thermal evaporation, ink jet printing, ultrasonic spraying,slot coating, gravure printing and lithography.

[0009] In another aspect of this embodiment, the second electrode is acounter electrode. The second electrode may include a metal in contactwith a metal salt, for example, silver in contact with silver chloride,silver in contact with silver bromide, silver in contact with silveriodide, mercury in contact with mercurous chloride, or mercury incontact with mercurous sulfate. The second electrode may also be areference electrode.

[0010] In another aspect of this embodiment, the electrochemical cellfurther includes a third electrode, such as a reference electrode. Thethird electrode may include a metal in contact with a metal salt, suchas silver in contact with silver chloride, silver in contact with silverbromide, silver in contact with silver iodide, mercury in contact withmercurous chloride, and mercury in contact with mercurous sulfate.

[0011] In another aspect of this embodiment, the reagent is capable ofoxidizing an analyte including an antioxidant. The reagent may includeferricyanide salts, dichromate salts, permanganate salts, vanadiumoxides, dichlorophenolindophenol, osmium bipyridine complexes, andquinones.

[0012] In another aspect of this embodiment, the reagent is capable ofreducing an analyte including an oxidant. The reagent may includeiodine, triiodide salts, ferrocyanide salts, ferrocene, Cu(NH₃)₄ ²⁺salts, and Co(NH₃)₆ ³⁺ salts.

[0013] In another aspect of this embodiment, the sensing chamber furtherincludes a buffer contained within the sensing chamber. The buffer isselected from the group consisting of phosphates, carbonates, alkalimetal salts of mellitic acid, and alkali metal salts of citric acid.

[0014] In another aspect of this embodiment, the device further includesa heating element. The heating element may include an electricallyresistive heating element or an exothermic substance contained withinthe sensing chamber, such as aluminum chloride, lithium chloride,lithium bromide, lithium iodide, lithium sulfate, magnesium chloride,magnesium bromide, magnesium iodide, magnesium sulfate, and mixturesthereof.

[0015] In another aspect of this embodiment, the sensing chamberincludes a support contained within the sensing chamber. Supports mayinclude mesh, nonwoven sheet, fibrous filler, macroporous membrane,sintered powder, and combinations thereof. One or both of the reagentand buffer may be contained within or supported on the support.

[0016] In another aspect of this embodiment, the second electrode ismounted in opposing relationship a distance of less than about 500microns from the first electrode, less than about 150 microns from thefirst electrode, or less than about 150 microns and greater than about50 microns from the first electrode.

[0017] In another aspect of this embodiment, the device further includesan interface for communication with a meter. The interface maycommunicate a voltage or a current.

[0018] In another aspect of this embodiment, the electrochemical cellincludes a thin layer electrochemical cell.

[0019] In a second embodiment of the present invention, a method fordetecting a presence or an absence of a redox reactive analyte in anaqueous sample is provided which includes providing a device fordetecting the presence or absence of an analyte in an aqueous sample,the device including an electrochemical cell having a sensing chamber, afirst electrode, a second electrode, an aperture for admitting thesample into the sensing chamber, and a reagent contained within thesensing chamber, wherein the electrochemical cell is designed to bedisposed of after use in a single experiment, and wherein the reagent iscapable of undergoing a redox reaction directly with the analyte togenerate an electrical signal indicative of the presence or absence ofthe analyte; providing an aqueous sample; allowing the sample to flowthrough the aperture and into the sensing chamber, such that the sensingchamber is substantially filled; and obtaining an electrochemicalmeasurement indicative of the presence or absence of analyte present inthe sample.

[0020] In one aspect of this embodiment, the electrochemical measurementis an amperometric measurement, a potentiometric measurement, acoulometric measurement, or a quantitative measurement.

[0021] In another aspect of this embodiment, the method includes thefurther step of heating the sample, wherein the heating step precedesthe step of obtaining the electrochemical measurement. Alternatively,the method may include the additional steps of heating the sample,wherein the heating step follows the step of obtaining anelectrochemical measurement; and thereafter obtaining a secondelectrochemical measurement indicative of the presence or absence of asecond analyte present in the sample.

[0022] In another aspect of this embodiment, the sensing chamber furtherincludes a buffer, for example, phosphate buffer, carbonate buffer,alkali metal salt of mellitic acid, and alkali metal salt of citricacid.

[0023] In a third aspect of the present invention, a method formeasuring sulfur dioxide in a sample of wine is provided, the sulfurdioxide having a free form and a bound form and being capable ofundergoing a redox reaction with a reagent, the redox reaction having areaction kinetics, wherein the method includes the steps of providing adevice, the device including an electrochemical cell having a sensingchamber, a first electrode, a second electrode, an aperture foradmitting the sample into the sensing chamber, and a reagent capable ofundergoing a redox reaction with sulfur dioxide, wherein theelectrochemical cell is designed to be disposed of after use in a singleexperiment; placing the sample of wine in the electrochemical cell,thereby initiating the redox reaction; and obtaining a firstelectrochemical measurement indicative of the level of sulfur dioxide infree form.

[0024] In one aspect of this embodiment, the method further includes thesteps of heating the sample of wine for a period of time sufficient forsulfur dioxide in bound form to react with the reagent, wherein theheating step is conducted after the step of obtaining a firstelectrochemical measurement; and thereafter obtaining a secondelectrochemical measurement indicative of the level sulfur dioxide infree form and in bound form combined. Alternatively, the method mayinclude the further steps of obtaining a second electrochemicalmeasurement indicative of the kinetics of reaction of the sulfur dioxidein bound form with the reagent, wherein the second electrochemicalmeasurement is obtained after the step of obtaining a firstelectrochemical measurement; and calculating the level of bound sulfurdioxide using the kinetics of reaction.

[0025] In a fourth aspect of the present invention, a method ofmanufacture of a device for detecting the presence or absence of a redoxreactive analyte in an aqueous sample is provided, the device includingan electrochemical cell having a sensing chamber, a first electrode, asecond electrode, an aperture for admitting the sample into the sensingchamber, and a reagent contained within the sensing chamber, wherein theelectrochemical cell is designed to be disposed of after use in a singleexperiment, and wherein the reagent is capable of undergoing a redoxreaction directly with the analyte to generate an electrical signalindicative of the presence or absence of the analyte, the methodincluding forming an aperture extending through a sheet of electricallyresistive material, the aperture defining a side wall of the sensingchamber; mounting a first layer having a first electrode to a first sideof the sheet and extending over the aperture, defining a first sensingchamber end wall, the first electrode facing the first side of thesheet; mounting a second layer having a second electrode to a secondside of the sheet and extending over the aperture defining a secondsensing chamber end wall in substantial overlying registration with thefirst layer, the second electrode facing the second side of the sheet,whereby the sheet and layers form a strip; forming an aperture in thestrip to permit entry of a sample into the sensing chamber; andproviding a reagent capable of undergoing a redox reaction directly withthe analyte, wherein the reagent is contained within the sensingchamber.

[0026] In one aspect of this embodiment, the method includes the furtherstep of providing a vent in the strip to permit escape of air displacedfrom the sensing chamber when sample fills the sensing chamber. Anotherfurther step includes mounting an electrically resistive heating elementto the strip.

[0027] In a further aspect of this embodiment, the aperture is of arectangular cross-section.

[0028] In a further aspect of this embodiment, at least one of theelectrodes includes a noble metal, for example, palladium, platinum, andsilver. At least one of the electrodes may be a sputter coated metaldeposit. The electrodes may be adhered to the sheet, for example, by anadhesive such as a heat activated adhesive, pressure sensitive adhesive,heat cured adhesive, chemically cured adhesive, hot melt adhesive, orhot flow adhesive.

[0029] In a further aspect of this embodiment, the method includesfurther steps such as providing an exothermic substance or buffercontained within the sensing chamber; printing the reagent or bufferonto at least one wall of the sensing chamber; or providing a supportsuch as mesh, fibrous filler, macroporous membrane, sintered powder, andcombinations thereof contained within the sensing chamber. The reagentmay be supported on or contained within the support.

[0030] In a further aspect of this embodiment, at least the sheet or oneof the layers of the device manufactured according to the method is apolymeric material selected from the group consisting of polyester,polystyrene, polycarbonate, polyolefin, and mixtures thereof.Alternatively, at least the sheet or one of the layers is polyethyleneterephthalate.

[0031] In a further aspect of this embodiment, the second electrode ismounted in opposing relationship a distance of less than about 500microns from the first electrode; less than about 150 microns from thefirst electrode; or less than about 150 microns and greater than about50 microns from the first electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 shows a plan view of an electrochemical cell.

[0033]FIG. 2 shows a cross-section view on line 10-10 of FIG. 1.

[0034]FIG. 3 shows an end-section view on line 11-11 of FIG. 1.

[0035]FIG. 4 shows schematically a heated electrochemical cell in across section taken longitudinally through the midline of the cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] The following description and examples illustrate a preferredembodiment of the present invention in detail. Those of skill in the artwill recognize that there are numerous variations and modifications ofthis invention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

[0037] The Sample and Analyte

[0038] In preferred embodiments, a method and device for measuringoxidant or antioxidant levels in fluid samples is provided. The methodand device are applicable to any oxidant or antioxidant that exists in ausefully representative concentration in a fluid sample. Antioxidantsthat may be analyzed include, for example, sulfur dioxide and ascorbicacid. Oxidants that may be analyzed include, for example, chlorine,bromine iodine, peroxides, hypochlorite, and ozone. Water insolubleoxidants or antioxidants may also be analyzed if an aqueous form can beprepared, e.g., by using a detergent to prepare an emulsion of the waterinsoluble redox reactive analyte.

[0039] Methods and devices for obtaining electrochemical measurements offluid samples are discussed further in copending U.S. patent applicationSer. No. 09/616,433, filed on Jul. 14, 2000, entitled “IMMUNOSENSOR,”copending U.S. patent application Ser. No. 09/616,512, filed on Jul. 14,2000, entitled “HEMOGLOBIN SENSOR,” and U.S. Pat. No. 6,444,115, issuedon Sep. 3, 2002, entitled “ELECTROCHEMICAL METHOD FOR MEASURING CHEMICALREACTION RATES,” each of which is incorporated herein by reference inits entirety.

[0040] The device and method may be used with any analyte-containingsample which is fluid and which is capable of solubilizing the redoxreagent to a sufficient extent. Typical samples include beverages suchas fruit and vegetable juice, carbonated beverages, drinking water,beer, wine, and spirits. However, it is not intended that the method belimited to comestible samples. If the sample is not in fluid form or isnot capable of solubilizing the redox reagent to a sufficient extent,the analyte contained within the sample may be extracted into a suitablefluid using extraction techniques well-known in the art. The sample maybe pre-treated prior to its introduction into the electrochemical cell.For example, pH may be adjusted to a desired level by means of a bufferor neutralizing agent, or a substance that renders interfering oxidantsor antioxidants nonreactive may be added. The sample may also bepreheated before introduction into the cell so as to accelerate the rateat which the redox reaction takes place.

[0041] The Electrochemical Cell

[0042] The electrochemical cell of present invention is disposable anddesigned for use in a single experiment. In a preferred embodiment, theelectrochemical cell is a thin layer sensor such as that disclosed inU.S. Pat. No. 5,942,102 (incorporated herein by reference in itsentirety). As herein used, the term “thin layer electrochemical cell”refers to a cell having closely spaced electrodes such that reactionproducts from the counter electrode arrive at the working electrode. Inpractice, the separation of electrodes in such a cell for measuringglucose in blood will be less than 500 microns, and preferably less than200 microns. A preferred embodiment of such an electrochemical cell isillustrated in FIGS. 1, 2, and 3. The cell illustrated in FIGS. 1, 2,and 3 includes a polyester core 4 having a circular aperture 8. Aperture8 defines a cylindrical cell side wall 12. Adhered to one side of core 4is a polyester sheet 1 having a sputter coating of palladium 2. Thesheet is adhered by means of an adhesive 3 to core 4 with palladium 2adjacent core 4 and covering aperture 8. A second polyester sheet 7having a second sputter coating of palladium 6 is adhered by means ofcontact adhesive 5 to the other side of core 4 and covering aperture 8.There is thereby defined a cell having cylindrical side wall 12 closedon each end by palladium metal 2, 6. The assembly is notched at 9 toprovide for a solution to be admitted to the cell or to be drawn in bywicking or capillary action and to allow air to escape. The metal films2, 6 are connected with suitable electrical connections or formationswhereby potentials may be applied and currently measured.

[0043] Such a thin layer electrochemical cell is prepared by firstforming an aperture extending through a sheet of electrically resistivematerial, the aperture defining a side wall of the electrochemical cell.Suitable electrically resistive materials, which may be used in thesheet containing the aperture, or in other layers in the cell, include,for example, materials such as polyesters, polystyrenes, polycarbonates,polyolefins, polyethylene terephthalate, mixtures thereof, and the like.In a preferred embodiment, the aperture in the sheet is rectangular,however other shapes, e.g., circular, may be used as well.

[0044] After the aperture is formed, a first thin electrode layer isthen mounted on one side of the sheet of electrically resistivematerial, extending over the aperture and forming an end wall. The layermay be adhered to the sheet, for example, by means of an adhesive.Suitable adhesives include, for example, heat activated adhesives,pressure sensitive adhesives, heat cured adhesives, chemically curedadhesives, hot melt adhesives, hot flow adhesives, and the like. Theelectrode layer is prepared by coating (e.g., by sputter coating) asheet of electrically resistive material with a suitable metal, forexample, palladium.

[0045] A second thin electrode layer is then mounted on the oppositeside of the electrically resistive material, also extending over theaperture, so as to form a second end wall. In a preferred embodiment,the electrode layers are mounted in opposing relationship at a distanceof less than about 1 millimeter, desirably less than about 800 microns,more desirably less that about 600, or preferably less than about 500microns, more preferably less than about 300 to 150 microns, morepreferably less than 150 microns, and most preferably between 25, 40,50, 100 and 150 microns. A second aperture or ingress is then providedfor liquid to enter the cell. Such an ingress can be provided by forminga notch along one edge of the device which extends through the electrodelayers and aperture. The electrode layers are provided with connectionmeans allowing the sensors to be placed in a measuring circuit.

[0046] Chemicals for use in the cell, such as redox reagents, buffers,and other substances, may be supported on the cell electrodes or walls,on one or more independent supports contained within cell, or may beself supporting. If the chemicals are to be supported on the cellelectrodes or walls, the chemicals may be applied by use of applicationtechniques well known in the art, such as ink jet printing, screenprinting, lithography, ultrasonic spraying, slot coating, gravureprinting, and the like. Suitable independent supports may include, butare not limited to, meshs, nonwoven sheets, fibrous fillers, macroporousmembranes, and sintered powders. The chemicals for use in the cell maybe supported on or contained within a support.

[0047] In a preferred embodiment, the materials used within the cell aswell as the materials used to construct the cell are in a form amenableto mass production, and the cells themselves are designed to be able tobe used for a single experiment then disposed of.

[0048] According to the present invention a disposable cell is one thatis inexpensive enough to produce that it is economically acceptable tobe used only for a single test. Secondly, that the cell may convenientlyonly be used for a single test. Inconveniently in this context meansthat steps such as washing and/or reloading of reagents would need to betaken to process the cell after a single use to render it suitable for asubsequent use.

[0049] Economically acceptable in this context means that the perceivedvalue of the result of the test to the user is the same or greater thanthe cost of the cell to purchase and use, the cell purchase price beingset by the cost of supplying the cell to the user plus an appropriatemark up. For many applications, this requires that the cells haverelatively low materials costs and simple fabrication processes. Forexample, the electrode materials of the cells should be inexpensive,such as carbon, or be used in sufficiently small amounts such thatexpensive materials may be used. Screen printing carbon or silver ink isa process suitable for forming electrodes with relatively inexpensivematerials. However, if it is desired to use electrode materials such asplatinum, palladium, gold or iridium, methods with better materialutilization, such as sputtering or evaporative vapor coating, are moresuitable as they may give extremely thin films. The substrate materialsfor the disposable cells also need to be inexpensive. Examples of suchinexpensive materials are polymers such as polyvinylchloride, polyimide,polyester and coated papers and cardboard.

[0050] Cell assembly methods also need to be amenable to massproduction. These methods include fabricating multiple cells on cardsand separating the card into individual strips subsequent to the mainassembly steps, and web fabrication where the cells are produced on acontinuous web, which is subsequently separated into individual strips.Card processes are most suitable when close spatial registration ofmultiple features is required for the fabrication and/or when stiff cellsubstrate materials are to be used. Web processes are most suitable whenthe down web registration of features is not as critical and flexiblewebs may be used.

[0051] The convenient single use requirement for the disposable cell isdesirable so that users are not tempted to try to reuse the cell andpossibly obtain an inaccurate test result. The single use requirementfor the cell may be stated in user instructions accompanying the cell.More preferably, the cell may also be fabricated such that using thecell more than once is difficult or not possible. This may beaccomplished, for example, by including reagents that are washed away orconsumed during the first test and so are not functional in a secondtest. Alternatively, the signal of the test may be examined forindications that reagents in the cell have already reacted, such as anabnormally high initial signal, and the test aborted. Another methodincludes providing a means for breaking electrical connections in thecell after the first test in a cell has been completed.

[0052] Cells for measuring antioxidants in the prior art do not satisfythese requirements for disposability. The cell disclosed by Richard J.Price et al. in Analyst, November 1991, Vol. 116, pages 1121-1123 uses asilver wire, a platinum wire and a platinum disc as the electrodes for acell measuring antioxidants in oil. Platinum wires are too expensive tobe used in a single use device in this application, and the cell isdesigned for continuous monitoring, not a single test. In U.S. Pat.No.5,518,590, Fang discloses another cell for measuring antioxidants inoil. This cell also uses platinum wire as an electrode and is alsodesigned for continuous use, namely, effectively conducting multipletests over time. This cell also requires a liquid or gel layercontaining a polar solvent. Such a device is not conducive to massfabrication and storage due to the need to contain the liquidcomponents, possibly over long periods, prior to use.

[0053] The Electrodes

[0054] At least one of the electrodes in the cell is a sensingelectrode, defined as an electrode sensitive to the amount of reducedredox agent in the antioxidant case or oxidized redox agent in theoxidant case. In the case of a potentiometric sensor wherein thepotential of the sensing electrode is indicative of the level of analytepresent, a second electrode acting as reference electrode is presentwhich acts to provide a reference potential.

[0055] In the case of an amperometric sensor wherein the sensingelectrode current is indicative of the level of analyte in the sample,at least one other electrode is present which functions as a counterelectrode to complete the electrical circuit. This second electrode mayalso function as a reference electrode. Alternatively, a separateelectrode may perform the function of a reference electrode.

[0056] Materials suitable for the sensing, counter, and referenceelectrodes must be compatible with the redox reagents present in thedevice. Compatible materials will not react chemically with the redoxreagent or any other substance present in the cell. Examples of suchsuitable materials include, but are not limited to, platinum, palladium,carbon, indium oxide, tin oxide, mixed indium/tin oxides, gold, silver,iridium and mixtures thereof. These materials may be formed intoelectrode structures by any suitable method, for example, by sputtering,vapor coating, screen printing, thermal evaporation or lithography. Inpreferred embodiments, the material is sputtered or screen printed toform the electrode structures.

[0057] Non-limiting examples of materials suitable for use in thereference electrode include metal/metal salt systems such as silver incontact with silver chloride, silver bromide or silver iodide, andmercury in contact mercurous chloride or mercurous sulfate. The metalmay be deposited by any suitable method and then brought into contactwith the appropriate metal salt. Suitable methods include, for example,electrolysis in a suitable salt solution or chemical oxidation. Suchmetal/metal salt systems provide better potential control inpotentiometric measurement methods than do single metal componentsystems. In a preferred embodiment, the metal/metal salt electrodesystems are used as a separate reference electrode in an amperometricsensor.

[0058] The Redox Reagent

[0059] Suitable redox reagents include those which are capable ofundergoing a redox reaction with the analyte of interest. Examples ofredox reagents suitable for use in analyzing antioxidant analytesinclude, but are not limited, to salts of ferricyanide, dichromate,osmium bipyridine complexes, vanadium oxides, and permanganate. Organicredox reagents such as dichlorophenolindophenol, and quinones are alsosuitable. In a preferred embodiment, the redox reagent for analyzing anantioxidant is ferricyanide. Examples of reagents suitable for use inanalyzing oxidant analytes include iodine and salts of triiodide,ferrocyanide, ferrocene, Cu(NH₃)₄ ²⁺, and Co(NH₃)₆ ³⁺. In a preferredembodiment, the redox reagent for measuring an oxidant is ferrocyanide.

[0060] The Buffer

[0061] Optionally, a buffer may be present along with the redox reagentin dried form in the electrochemical cell. If a buffer is used, it ispresent in an amount such that the resulting pH level is suitable foradjusting the oxidizing (or reducing) potential of the redox reagent toa level suitable for oxidizing (or reducing) the analytes of interestbut not other species that it is not desired to detect. The buffer ispresent in a sufficient amount so as to substantially maintain the pH ofthe sample at the desired level during the test. Examples of bufferssuitable for use include phosphates, carbonates, alkali metal salts ofmellitic acid, and alkali metal salts of citric acid. The choice ofbuffer will depend on the desired pH. The buffer is selected so as notto react with the redox reagent. Alkali buffers are preferred for use inconjunction with carbonated beverages.

[0062] Other Substances Present Within The Cell

[0063] In addition to redox reagents and buffers, other substances mayalso be present within the cell. Such substances include, for example,viscosity enhancers and low molecular weight polymers. Hydrophilicsubstances may also be contained within the cell, such as polyethyleneglycol, polyacrylic acid, dextran, and surfactants such as thosemarketed by Rohm & Haas Company of Philadelphia, Pa., under the tradename Triton™ or by ICI Americas Inc. of Wilmington, Del., under thetrade name Tween™. Such substances may enhance the fill rate of thecell, provide a more stable measurement, and inhibit evaporation insmall volume samples.

[0064] Method for Measuring Analyte Concentration

[0065] In measuring an antioxidant or oxidant analyte present in asample, the sample is introduced into the sensor cell, whereupon thesample dissolves the dried reagents present in the cell. The redoxreagent then reacts with any antioxidants or oxidants of interestpresent in the sample to form the reduced or oxidized form of the redoxreagent. In the case of a potentiometric sensor, the resulting ratio ofoxidized to reduced form of the redox reagent fixes the potential of thesensing electrode relative to the reference electrode. This potential isthen used as a measure of the concentration of the analyte originally inthe sample.

[0066] In a preferred embodiment, the sensing cell is operated as anamperometric sensor. According to this embodiment, the reduced (oroxidized) redox reagent formed by reaction with the analytes of choiceis electrochemically oxidized (or reduced) at the sensing electrode. Thecurrent resulting from this electrochemical reaction is then used tomeasure the concentration of analytes originally in the sample. In otherembodiments, the sensor is operated in potentiometric or coulometricmode.

[0067] The cell's electrodes are used to produce an electrical signal,i.e., a voltage or current, readable by an attached meter. In apreferred embodiment, an interface for connecting the cell to the meteris provided. The meter may display the measurement in a visual, audio orother form, or may store the measurement in electronic form.

[0068] Heating the Sample

[0069] Certain oxidant or antioxidant analytes are slow to react withthe redox reagent. To accelerate the reaction, and thus reduce the timerequired to obtain the measurement, the sample may be heated. In apreferred embodiment, a means for heating the sample is provided in thedisposable electrochemical sensor device.

[0070] Two suitable means of heating the cell are described inWO99/46585 (incorporated herein by reference in its entirety).WO99/46585 discloses a method for determining the concentration of ananalyte in a sample wherein the sample is heated and the concentrationof the analyte (or species representative of the analyte) is measured ata predetermined point on a reaction profile (defined as the relationshipof one reaction variable to another) by temperature independent means.The sample may be heated either by an exothermic reaction produced uponcontact of the sample with a suitable reagent or reagents or the samplemay be heated electrically by means of a current applied to resistiveelements associated with the cell.

[0071] One method of heating the sample via exothermic reaction involvesplacing in the electrochemical cell a reagent that liberates heat oncontact with the sample. Examples of such reagents include salts whichgive out heat when they dissolve, such as aluminum chloride, lithiumhalide salts, lithium sulfate, magnesium halide salts and magnesiumsulfate. The reagent or reagents used to liberate heat must notadversely affect the function of the other active elements in the cell,such as by corroding electrode materials, reacting with the analyte soas to affect its response, or adversely interacting with other reagentspresent.

[0072] When the sample is to be heated electrically, the electrochemicalcell may be equipped with an electrically resistive element. FIG. 4shows a preferred embodiment of an electrochemical sensor as describedin WO99/46585. The sensor comprises a nonconducting substrate 21,bearing a first electrode 22, a separator layer 23 having a circularaperture 30 punched out which defines a circular cell wall 30. The firstelectrode 22 defines one end of the cell, the other end being defined bythe second electrode layer 24, which is carried by a secondnonconducting layer 25. A metal foil layer 26, provides electricalcontact to a resistive bridge 29 formed in the second nonconductinglayer 25. An insulating layer 27 provides insulation against heat lossthrough the metal foil layer 26. An aperture 28 is formed in insulatinglayer 27 to allow access for electrical connection to foil 26.

[0073] In preferred embodiments, resistive elements may be prepared byimpregnating one or more of the nonconducting layers carrying anelectrode layer with a substance such as carbon particles. Thenonconducting layers may include such materials as plastic or rubber.The impregnated rubber or plastic layer forms a resistive bridge betweenthe electrode of the electrochemical cell and the metal foil layer. Whena potential is applied across the resistive element, heat is generatedin the impregnated rubber or plastic layer, which in turn heats thesample in the electrochemical cell. Alternatively, at least two lowresistance tracks joined by a high resistance track can be formed on anexternal face of the sensor. In such an embodiment, the low resistancetracks serve to make contact with the meter and the high resistancetrack forms the electrically resistive element.

[0074] Multiple Cell Devices

[0075] In certain situations, it may be desirable to measure more thanone oxidant and/or antioxidant analyte in a sample. This may beaccomplished by using an array of two or more electrochemical cells asdescribed above. Each cell contains a redox reagent suited for use withone of the analytes present in the sample. Each cell is also equippedwith buffers or heating means, if required for that particular analyte.Such an array of cells may be used not only to determine theconcentration of known analytes of interest, but may also be used toscreen a sample of unknown analyte composition for the presence orabsence of a variety of analytes.

[0076] Various embodiments of a cell array are contemplated. In oneembodiment, cell construction techniques as described above are used tofabricate a device having multiple sensing chambers and electrodes butsharing one or more layers of insulating material. In anotherembodiment, two or more electrochemical cells as described above areadhered together, either directly to each other or to a separate supportmaterial. Alternatively, two or more cells as described above, butcontaining different reagents, may be packaged together in a kitsuitable for use in a particular application, i.e., a analysis of asample containing multiple analytes or different forms of the sameanalyte.

[0077] Analysis of Sulfur Dioxide in Wine

[0078] One example of an analysis wherein it is useful to heat thesample is the measurement of sulfur dioxide in wine. Sulfur dioxide inwine functions as an antioxidant and is typically present in two forms:the free form and the bound form. The free form is more quickly oxidizedby the redox reagent in the sensor than is the bound form. It isnormally desirable to measure both the free and bound forms of sulfurdioxide in wine. To measure both forms, a heating means is included inthe electrochemical cell. A sample of the wine is placed in the sensingcavity, whereupon the redox reagent present reacts quickly with the freesulfur dioxide to produce a sensor signal. This signal is analyzed andthen heat is applied to the sample via the heating means. In a preferredembodiment, heating is applied with a slow rise in temperature so as toavoid excessive evaporation of the sample. After a suitable period oftime at elevated temperature, the bound sulfur dioxide reacts with theredox reagent, thereby producing a second sensor signal. From these twosignals the free concentration and total concentration of sulfur dioxidein the sample are obtained, and thus, by difference, are the free andbound form concentrations obtained. While this two-step method isbeneficial for obtaining the concentration of the free and bound formsof sulfur dioxide in wine, the invention also contemplates other usesfor such a method. For example, a two (or more) step method may be usedfor analyzing suitable samples containing an analyte having two or moreforms with different reaction kinetics, or samples containing two ormore different analytes each having different reaction kinetics.

[0079] The above description discloses several methods and materials ofthe present invention. This invention is susceptible to modifications inthe methods and materials, as well as alterations in the fabricationmethods and equipment. Such modifications will become apparent to thoseskilled in the art from a consideration of this disclosure or practiceof the invention disclosed herein. Consequently, it is not intended thatthis invention be limited to the specific embodiments disclosed herein,but that it cover all modifications and alternatives coming within thetrue scope and spirit of the invention as embodied in the attachedclaims.

What is claimed is:
 1. A device for detecting a presence or an absenceof a redox reactive analyte in an aqueous sample, the device comprisingan electrochemical cell having a sensing chamber, a first electrode, asecond electrode, an aperture for admitting the sample into the sensingchamber, and a reagent contained within the sensing chamber, wherein thedevice contains a quantity of the reagent sufficient for only a singletest, and wherein the reagent is capable of undergoing a redox reactiondirectly with the analyte to generate an electrical signal indicative ofthe presence or absence of the analyte.
 2. The device of claim 1,wherein the first electrode comprises a sensing electrode.
 3. The deviceof claim 1, wherein the first electrode comprises a material selectedfrom the group consisting of platinum, palladium, carbon, indium oxide,tin oxide, gold, iridium, copper, steel, silver, and mixtures thereof.4. The device of claim 1, wherein the second electrode comprises acounter electrode.
 5. The device of claim 1, wherein the secondelectrode comprises a metal in contact with a metal salt.
 6. The deviceof claim 5, wherein the metal in contact with a metal salt is selectedfrom the group consisting of silver in contact with silver chloride,silver in contact with silver bromide, silver in contact with silveriodide, mercury in contact with mercurous chloride, and mercury incontact with mercurous sulfate.
 7. The device of claim 1, theelectrochemical cell further comprising a reference electrode.
 8. Thedevice of claim 1, wherein the reagent is capable of oxidizing ananalyte comprising an antioxidant.
 9. The device of claim 8, wherein thereagent is selected from the group consisting of ferricyanide salts,dichromate salts, permanganate salts, vanadium oxides,dichlorophenolindophenol, osmium bipyridine complexes, and quinones. 10.The device of claim 1, wherein the reagent is capable of reducing ananalyte comprising an oxidant.
 11. The device of claim 10, wherein thereagent is selected from the group consisting of iodine, triiodidesalts, ferrocyanide salts, ferrocene, Cu(NH₃)₄ ²⁺ salts, and Co(NH₃)₆ ³⁺salts.
 12. The device of claim 1, the sensing chamber further comprisinga buffer, wherein the buffer is contained within the sensing chamber.13. The device of claim 12, wherein the buffer is selected from thegroup consisting of phosphates, carbonates, alkali metal salts ofmellitic acid, and alkali metal salts of citric acid.
 14. The device ofclaim 1, further comprising a heating element.
 15. The device of claim14, wherein the heating element is an electrically resistive heatingelement.
 16. The device of claim 14, wherein the heating element is anexothermic substance contained within the sensing chamber.
 17. Thedevice of claim 1, wherein the second electrode is mounted in opposingrelationship a distance of less than about 150 microns from the firstelectrode.
 18. The device of claim 1, further comprising an interfacefor communication with a meter.
 19. The device of claim 18, wherein theinterface communicates a voltage or a current.
 20. The device of claim1, wherein the electrochemical cell comprises a thin layerelectrochemical cell.